Blood online
Home About Blood Authors Subscriptions Permission Advertising Public Access contact us
 

 
Advanced
Current Issue
First Edition
Archives
Submit to Blood
Search
American Society of Hematology
Meeting Abstracts
Email Alerts
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Skarpidi, E.
Right arrow Articles by Li, Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skarpidi, E.
Right arrow Articles by Li, Q.
Related Collections
Right arrow Red Cells
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

arrow to previous article Previous Article  |  Table of Contents  |  Next Article next article arrow

Blood, Vol. 92 No. 9 (November 1), 1998: pp. 3416-3421

Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

By E. Skarpidi, G. Vassilopoulos, G. Stamatoyannopoulos, and Q. Li

From the Division of Medical Genetics, University of Washington, Seattle, WA.


    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

To examine whether transfer of gamma  globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma  globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (µLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the µLCR(-201)Agamma and µLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma  globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.

© 1998 by The American Society of Hematology.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

ANALYSIS OF THE cis control of globin gene expression has greatly benefited from studies of transgenic mice.1 A major advance in this field has been the discovery of the locus control region (LCR), the major regulatory element of the beta  locus, which resides 6 to 20 kb upstream of the epsilon  globin gene.2 The LCR activates the beta  locus chromatin domain, it serves as a chromatin "insulator" by protecting globin transgenes from the effects of integration in inactive chromatin, and it is a powerful enhancer of globin gene expression.1,2 The discovery of the LCR triggered the extensive use of transgenic mice for the analysis of globin gene expression. Studies in transgenic mice have provided information on the function of the individual DNase I hypersensitive sites of the LCR,3-14 have shown that competition between genes for interaction with the LCR plays a role in the control of globin gene switching,15,16 have led to the discovery of cis elements involved in epsilon 17,18 and gamma 19,20 gene silencing, and have provided insights on the function of downstream regulatory elements21-23 and on the interaction between transcriptional factors and globin gene regulatory motifs.24

Another experimental system for analysis of the cis control of globin gene expression uses transfections of human genes into established erythroleukemia cell lines. Mouse erythroleukemia (MEL) cells, although arrested at a differentiation stage before globin gene transcription, they are capable of chemically induced erythroid maturation during which high levels of human globin gene expression are observed. Thus, they have been used for studies of induction of globin gene expression by various compounds and in the analysis of regulatory elements of the beta  globin locus.25-30 Typically, in these studies, the cells are transfected either transiently or stably with recombinant human globin genes and are induced to terminal maturation by exposure to various chemical agents like dimethyl sulfoxide (DMSO) or hexamethylane-bis-acetamide (HMBA). Measurements of globin gene expression before and after MEL cell induction provide information on the regulatory role of the sequences contained in the transferred gene.

MEL cells are an adult erythroleukemia cell line characterized by synthesis of adult beta major and beta minor globin chains; there is no transcription of murine embryonic (beta h1 and epsilon y) globin genes. There is only adult human globin production when the chromosomal human globin genes from adult erythroid cells or from lymphoblasts or fibroblasts are transferred into the MEL cells by cell fusion.31,32 Although MEL cells are derived from the adult hematopoietic lineage, their transcriptional environment is permissive of human gamma  globin gene expression. Thus, when the chromosome 11 of human fetal erythroblasts is transferred into MEL cells, the human gamma  globin genes continue to be expressed.32 These MELxhuman fetal erythroid cell hybrids initially express predominantly or exclusively human gamma  globin and over time in culture they switch to predominantly or exclusively beta  globin expression.32 gamma  Gene expression also continues when human chromosomes containing either the -117Agamma hereditary persistence of fetal hemoglobin (HPFH) gene or a Ggamma Agamma deletion HPFH gene are transferred into MEL cells by cell fusion.33

We were interested to assess whether MEL cells can substitute the transgenic mice for the analysis of the cis elements, which regulate human gamma  globin gene expression. To obtain this information, we analyzed gamma  gene expression in MEL cells stably transfected with various Agamma globin gene promoter truncation constructs, which have been previously characterized in transgenic mice.19 Our results suggest that MEL cells can be used for the analysis of cis elements whose mutations produce severe deficiency in Agamma globin mRNA. This cell line is less useful for the study of cis elements that have moderate effects on gamma  gene expression or for studies of the effects of position of integration on the expression of transferred gamma  globin genes.

    MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Recombinant constructs.   Four constructs, which contain a 2.5-kb µLCR cassette linked to various Agamma gene promoter truncations, were used.19 These Agamma gene recombinants have the same 3' end at the HindIII position 530 bp downstream to the polyadenylation site of the human Agamma gene, while they differ regarding their 5' ends. The 5' end of the µLCR(-141)Agamma construct is in the Nco I site at position -141. The gamma  promoter of the µLCR(-201)Agamma construct extends to the Apa I site at position -201, while in the µLCR(-382)Agamma and µLCR(-730)Agamma construct, the promoter extends to the StuI and SspI sites at positions -382 and -730, respectively.

MEL cell culture and DNA transfection.   MEL 585 cells were maintained in RPMI 1640 medium with 10% fetal calf serum. Cells, 10 × 106, were cotransfected with 1 µg TKneo and a 3-fold to 10-fold molar excess of linearized Agamma gene fragments by electroporation at 0.6 kV, 1 µF using a Bio-Rad Gene Pulser set (Bio-Rad Laboratories, Hercules, CA). After electroporation, individual cell populations were allowed to grow without selection for 48 hours, then selection was applied using medium supplemented with 700 µg/mL G418. After about 7 to 10 days in culture, populations of rapidly growing cells are observed. Individual clones for each of the transfected constructs were produced from a pool using limiting dilution assay. It is known that prolonged culture of a pool, even a large one, may result in the predominance of one or two clones. For this reason, the clones were generated shortly after the production of the cell pools to ensure the presence of different cell clones. This has also been confirmed by the Southern analysis, which showed the presence of various copy numbers of the recombinant in the clones studied. Cells were induced with 3 mmol/L HMBA/10 µmol/L hemin for 3 days before harvesting for RNA analysis.

In another set of experiments after electroporation, the cells were plated in 48-well plates at approximately 105 cells per well in selection medium containing 700 µg/mL G418. Cell concentrations were such as to yield <= 1 positive clone per well based on estimated transfection efficiency. After approximately 14 days, individual G418-resistant clones were picked and pooled for the generation of cell populations with defined number of clones (10 or 50). The cells were subsequently expanded, induced to differentiate as described above, for 3 days, and harvested for RNA analysis.

Copy number determination.   Genomic DNA was isolated from the pooled cells by standard procedures. DNA samples were digested overnight with EcoRI restriction enzyme. About 2 to 10 µg of the digested DNA (as determined by fluorometry) was resolved by electrophoresis in 1× TAE buffer over 1% agarose and transferred onto nylon filter. The filter was subsequently hybridized with a 0.8-kb Agamma globin probe and signals were quantitated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Copy numbers were determined by comparing the signals from a given MEL cell sample with those of human genomic (ie, two-copy) DNA (Promega, Madison, WI).

RNA analysis and quantitation.   Total cellular RNA was isolated by the acid-phenol method described previously.12 Human Agamma and murine alpha  globin mRNAs were quantitated by RNase protection assay. Antisense RNA probes were synthesized from linearized DNA templates using T7 polymerase. Probe pT7Agamma detects a 170-bp protected fragment derived from exon 2 of the Agamma mRNA. Probe pT7Malpha , directed against murine alpha  globin mRNA, was derived from pSP6Malpha through the replacement of the SP6 promoter with the T7 promoter. Probe pTRIRNA 18s (Ambion, Austin, TX) was used to identify an 80-bp protected fragment.

The levels of human Agamma and murine alpha  globin mRNA were quantitated by Phosphorimager analysis. To maximize the accuracy of RNase protection assays, at least two independent assays were performed for measurement of globin mRNA levels in each pool or clone of MEL cells. Agamma mRNA levels were corrected for RNA loading using the levels of 18S RNA as a standard.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Globin transgene expression in MEL cell clones is strongly influenced by the position of integration.   Constructs which contain globin gene sequences that participate in the interaction between globin genes and the LCR display copy number-dependent, position of integration-independent expression of globin genes in transgenic mice. Cis elements, which are located in the Agamma globin gene promoter and either confer position-independent Agamma globin gene expression [µLCR(-201) or µLCR(-382) Agamma construct] or position-dependent expression [µLCR(-730) or µLCR(-1350) Agamma construct], have been previously identified by studies in transgenic mice.19 To examine whether MEL cells can be used for the study of elements involved in LCR/globin gene interaction, we measured Agamma globin mRNA levels among individual MEL cell clones containing the µLCR(-201)Agamma , µLCR(-382)Agamma , and µLCR(-730)Agamma constructs. Each MEL cell clone was induced to terminal maturation with HMBA and hemin and Agamma mRNA levels in induced cells were measured by quantitative RNase protection assay. Agamma mRNA levels were expressed as percentage of the murine alpha  globin mRNA after correction for the number of copies of the µLCRAgamma recombinant contained in each clone.

As shown in Fig 1, a striking degree of variation in Agamma gene expression is characteristic of the clones carrying these constructs. Large variation in Agamma gene expression is not expected for the µLCR(-201)Agamma and the µLCR(-382)Agamma constructs, which in transgenic mice are characterized by copy number-dependent, position-independent expression. Agamma mRNA levels among the µLCR(-201)Agamma and µLCR(-382)Agamma MEL cell clones ranged 15.5-fold (from 14.4% to 223.1% of murine alpha  mRNA per copy) and 17.6-fold (from 3.0% to 52.8% per copy), respectively. While in transgenic mice, there is excellent correlation (r = .95, P <=  .0001) between the Agamma mRNA levels and the number of copies of the integrated µLCR(-201)Agamma and µLCR(-382)Agamma constructs (Fig 2A), there is no correlation (r = .28, P = .18) between the Agamma mRNA levels and the number of copies of the integrated µLCR(-201)Agamma and µLCR(-382)Agamma constructs in MEL cell clones (Fig 2B). These results indicate that globin gene constructs, which are not influenced by the position of integration in transgenic mice, become sensitive to position effects when they are transferred into MEL cells.


View larger version (13K):
[in this window]
[in a new window]
  		Fig 1. Agamma gene expression in clones of MEL cells carrying the µLCR(-201)Agamma , µLCR(-382)Agamma , and µLCR(-730)Agamma constructs. Notice the striking degree of variation in Agamma gene expression among clones carrying the same construct. Horizontal lines represent mean Agamma mRNA values.


View larger version (16K):
[in this window]
[in a new window]
  		Fig 2. Correlation between total Agamma mRNA levels and transgene copy numbers in transgenic mouse lines (A) or MEL cell clones (B) carrying the µLCR(-201)Agamma and µLCR(-382)Agamma constructs. The excellent correlation observed in transgenic mice (r = .96, P < .0001) does not exist in MEL cell clones (r = .28, P = .18).

Comparison of Agamma gene expression in MEL cell pools and transgenic mice.   Four recombinants carrying Agamma gene promoter truncations [µLCR(-141)Agamma , µLCR(-201)Agamma , µLCR (-382)Agamma , and µLCR(-730)Agamma ] were transferred to MEL cells and pools of stably transfected cells were generated and induced to terminal maturation with HMBA and hemin. Levels of Agamma mRNA in uninduced and induced cells were expressed as percentage of the murine alpha  mRNA after correction for the average number of copies of the µLCRAgamma recombinant contained in each pool and for the number of copies of the endogenous murine alpha  globin genes. Results are summarized in Table 1.

 
View this table:
[in this window] [in a new window]
  		Table 1. gamma Globin Gene Expression in MEL Cell Pools Transfected With Four Agamma Promoter Truncation Mutants

The µLCR(-141)Agamma pools display the lowest level of Agamma transgene expression (range from 1.6% to 4.8% of murine alpha  mRNA per copy; mean value, 2.7%). In the µLCR(-201)Agamma pools, the levels of Agamma mRNA were about one order of magnitude higher compared with the (-141)Agamma pools (mean value, 29.9%; range from 11.7% to 73% of murine alpha ). The µLCR(-201)Agamma , µLCR(-382)Agamma , and µLCR(-730)Agamma pools had similar mean Agamma mRNA levels (29.9%, 29.4%, 30.3% of murine alpha  mRNA per copy, respectively). Agamma mRNA levels varied by 6.2-fold among the µLCR(-201)Agamma pools, 2.4-fold among the µLCR(-382)Agamma pools, and 9.9-fold among the µLCR(-730)Agamma pools. The variation in Agamma mRNA levels among pools was significantly lower than among clones carrying the same contructs.

In Fig 3, we compare Agamma mRNA levels in the MEL cell pools and transgenic mice. Each data point in the transgenic mice columns corresponds to the mean value of Agamma mRNA in several adult animals of each transgenic line. The overall pattern of globin gene expression between MEL cell pools resembles that in transgenic mice. In both MEL cells and transgenic mice, disruption of the Agamma gene CACCC box, as in the µLCR(-141)Agamma construct, severely affects Agamma gene expression. Addition of 5' sequences, as in the µLCR(-201)Agamma or the µLCR(-382)Agamma constructs, restores Agamma gene expression to about 30% of murine alpha . Several differences in globin gene expression between transgenic mice and MEL cell pools are also noted. Thus, the µLCR(-141)Agamma recombinant, which in the adult transgenic mice shows barely measurable Agamma mRNA levels, displays considerable expression in MEL cells. The mean Agamma mRNA levels in MEL cell pools carrying the µLCR(-201)Agamma , µLCR(-382)Agamma , and µLCR(-730)Agamma constructs are almost twofold higher than those in adult mice carrying the same transgenes. While Agamma mRNA expression in the µLCR(-201)Agamma transgenic mice is position-independent, as shown by the small degree of variation of the Agamma mRNA levels among transgenic lines, Agamma mRNA levels in the µLCR(-201)Agamma MEL cell pools display about sixfold variation, indicating that Agamma gene expression is position-dependent (Fig 3B).


View larger version (8K):
[in this window]
[in a new window]
  		Fig 3. Agamma gene expression for four Agamma promoter truncation constructs in MEL cell pools and transgenic mice. Levels of Agamma mRNA are expressed as percentage of murine alpha mRNA per copy of transgene and copy of murine alpha gene. Horizontal lines represent mean Agamma mRNA values. Notice that in panels A1 and A2, mRNA levels for the µLCR(-141)Agamma construct are plotted in a different scale to demonstrate the observed difference in expression between cells and mice.

The variation in Agamma /alpha mRNA levels among MEL cell pools could be caused by the presence of small numbers of clones comprising each pool. Pools containing few clones are expected to be randomly affected by the large variation of Agamma globin gene expression among clones. When the pools are comprised of a large number of clones, any position effects on Agamma globin gene expression in the clones should be "averaged," resulting in smaller variation in Agamma /alpha gene expression among pools. To examine this possibility, MEL cell pools carrying the µLCR(-382)Agamma construct consisting of either 10 or 50 single clones each were generated and globin mRNA levels were measured in induced cells. As shown in Table 2, pools of 10 clones displayed a 14.1-fold variation in the level of Agamma mRNA (range from 1.5% to 21.1% of murine alpha  mRNA per copy). Agamma mRNA levels among pools composed of 50 clones varied by only 2.2-fold (from 15.4% to 33.6% of murine alpha ). These data show that a significant number of individual clones (at least 50) must be present in each MEL cell pool to adjust for the effects of position of integration on the expression of the transgene.

 
View this table:
[in this window] [in a new window]
  		Table 2. gamma Globin Gene Expression in MEL Cell Pools Containing 10 or 50 Individual Clones Carrying the µLCR(-382)Agamma Construct

Coordinate induction of exogenous and endogenous globin genes.   For studies of globin genes transferred into MEL cells, the cells are cultured in the presence of HMBA and hemin for 72 hours during which terminal maturation occurs and hemoglobin is produced. The process of MEL cell induction is empirical and experiments may vary in the degree of MEL cell induction, as judged by the degree of hemoglobinization. When the levels of transgene mRNA are expressed as percent of the endogenous murine alpha  gene, variation in MEL cell inducibility between experiments could contribute to variation in expression if the human transgene and the endogenous gene are not induced in a coordinate fashion. Discordant human Agamma and murine alpha  globin gene induction will result in random variation of human/mouse mRNA ratios among MEL cells carrying the same construct.

To examine whether induction of the human Agamma and murine alpha  genes is coordinate, we calculated the ratio of Agamma and alpha  mRNAs in induced (I) and uninduced (U) MEL cells. There was considerable variation among MEL cell pools in I/U Agamma mRNA ratios (up to 4.9-fold) and an even larger variation in I/U alpha  mRNA ratios (10.4-fold). However, as shown in Fig 4, there is a statistically significant correlation between the I/U Agamma mRNA and the I/U alpha  mRNA ratios (r = 0.74, P < .0001), suggesting that the exogenous Agamma and the endogenous alpha  globin genes are induced in a coordinate fashion. Therefore, random differences in the degree of Agamma and alpha  globin gene induction do not significantly contribute to the observed variation of Agamma /alpha mRNA levels.


View larger version (14K):
[in this window]
[in a new window]
  		Fig 4. Regression analysis of the induced/uninduced Agamma and alpha mRNA ratios. A statistically significant correlation is observed (r = .74, P < .0001), indicating that the exogenous and endogenous genes are induced coordinately.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Transgenic mice have been extensively used for elucidation of the cis elements that control globin gene expression,3-23,34-37 but studies with this model system are expensive and time-consuming. Using transgenic mice, we have previously obtained evidence that the Agamma promoter region between nucleotides -382 and -730 relative to the cap site harbors a position-dependent gamma  gene silencer.19 To further delineate this element and demarcate the sequences that contain the putative gamma  gene silencer, the function of a large number of Agamma gene promoter mutations needs to be determined. To test whether stable transfections of MEL cells could be used for that purpose, we transfected MEL cells with Agamma globin gene constructs whose function has been well defined in transgenic mice and asked whether the results obtained in transgenic mice can be reproduced in MEL cell clones and/or pools. We found that the MEL cell system is not useful for studies of elements which protect globin gene expression from position effects. Typically, such studies require clonal analysis and measurement of gene expression per copy of the transgene. Copy number-dependent expression indicates that gene expression is not influenced by the position of integration of the transgene, while copy number-independent expression indicates that the transgene is sensitive to position effects. As we have shown in the present study, constructs that in transgenic mice display position-independent, copy number-dependent expression, become sensitive to position effects in MEL cells. Therefore, it is unlikely that MEL cell transfectants can provide useful information on globin gene sequences, which participate in LCR-globin gene interactions. Another implication of our observations concerns the evaluation of globin gene retroviral vectors. Such retroviral vectors developed for the purpose of gene therapy for hemoglobinopathies, usually contain sequences of the human beta  or gamma  globin genes linked to various LCR cassettes.38-43 Studies in transduced MEL cell clones are performed to test whether the LCR cassette contained in the retroviral vector enhances globin gene expression and protects it from position effects. Our results suggest that variation in globin gene expression among transduced MEL cell clones does not necessarily signify that a retroviral vector is sensitive to position effects.

The great majority of the clones used in this study contained multiple copies of integrants. It has been suggested that the presence of multiple transgene copies may adversely affect the level of transgene expression.26 Although no correlation between transgene copy number and expression per copy was observed in this study, it has been recently reported44 that single copy transgenes demonstrate consistent and reproducible expression in stably transfected MEL cells. It is possible that the presence of multiple integrants accounts for the observed variation in Agamma globin gene expression among MEL cell clones.

The comparison of globin gene expression between MEL cell pools and transgenic mice shows that MEL cell pools may be useful in identifying cis elements whose mutations severely affect gamma globin gene expression. Thus, the deleterious effect of CACC box disruption [in the µLCR(-141)Agamma pools] on Agamma gene expression and the restoration of expression upon addition of upstream sequences [in the µLCR(-201)Agamma pools], which were demonstrated in transgenic mice, were also observed in MEL cell pools. However, studies in MEL cells pools cannot identify elements that moderately affect globin gene expression. This is apparent from the findings in transgenic mice and MEL cell pools carrying the µLCR(-730)Agamma construct. In transgenic mice, this construct displayed 500-fold variation among lines and significant decrease in expression in the adult mice, showing the presence of a position-dependent gamma globin gene silencer. Such effects were not observed in the MEL cell pools transfected with this construct.

    FOOTNOTES

   Submitted August 18, 1997; accepted June 19, 1998.
   Supported by Grants No. HL46557, HL53750, and DK45365 from the National Institutes of Health, an International Fogarty Fellowship Award to G.V., and a Cooley's Anemia Foundation Award to E.S.
   Address reprint requests to G. Stamatoyannopoulos, MD, Medical Genetics, Box 357720, University of Washington, Seattle, WA 98195; e-mail: gstam{at}u.washington.edu.
   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

1. Nienhuis AW: Hemoglobin switching , in Stamatoyannopoulos G, Nienhuis AW, Majerus P, Varmus H (eds): The Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders , 1994 , p 107

2. Grosveld F, Blom van Assendelft G, Greaves DR, Kollias G: Position-independent, high-level expression of the human beta -globin gene in transgenic mice. Cell 51:975, 1987[Medline] [Order article via Infotrieve]

3. Collis P, Antoniou M, Grosveld F: Definition of the minimal requirements within the human beta -globin gene and the dominant control region for high level expression. EMBO J 9:233, 1990[Medline] [Order article via Infotrieve]

4. Talbot D, Philipsen S, Fraser P, Grosveld F: Detailed analysis of the site 3 region of the human beta -globin dominant control region. EMBO J 9:2169, 1990[Medline] [Order article via Infotrieve]

5. Philipsen S, Talbot D, Fraser P, Grosveld F: The beta -globin dominant control region: Hypersensitive site 2. EMBO J 9:2159, 1990[Medline] [Order article via Infotrieve]

6. Philipsen S, Pruzina S, Grosveld F: The minimal requirements for activity in transgenic mice of hypersensitive site 3 of the beta  globin locus control region. EMBO J 12:1077, 1993[Medline] [Order article via Infotrieve]

7. Pruzina S, Hanscombe O, Whyatt D, Grosveld F, Philipsen S: Hypersensitive site 4 of the human beta  globin locus control region. Nucleic Acids Res 19:1413, 1991[Abstract/Free Full Text]

8. Fraser P, Hurst J, Collis P, Grosveld F: DNaseI hypersensitive sites 1, 2 and 3 of the human beta -globin dominant control region direct position-independent expression. Nucleic Acids Res 18:3503, 1990[Abstract/Free Full Text]

9. Ryan TM, Behringer RB, Martin NC, Townes TM, Palmiter RD, Brinster RL: A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta -globin gene expression in transgenic mice. Genes Dev 3:314, 1989[Abstract/Free Full Text]

10. Liu D, Chang JC, Moi P, Liu W, Kan YW, Curtin PT: Dissection of the enhancer activity of beta -globin 5' DNase I-hypersensitive site 2 in transgenic mice. Proc Natl Acad Sci USA 89:3899, 1992[Abstract/Free Full Text]

11. Morley BJ, Abbott CA, Sharpe JA, Lida J, Chan-Thomas PS, Wood WG: A single beta -globin locus control region element (5' hypersensitive site 2) is sufficient for developmental regulation of human globin genes in transgenic mice. Mol Cell Biol 12:2057, 1992[Abstract/Free Full Text]

12. Li Q, Stamatoyannopoulos J: Position independence and proper developmental control of gamma -globin gene expression require both a 5' locus control region and a downstream sequence element. Mol Cell Biol 14:6087, 1994[Abstract/Free Full Text]

13. Bungert J, Dave U, Lim K-C, Lieuw KH, Shavit JA, Liu Q, Engel JD: Synergistic regulation of human beta -globin gene switching by locus control region elements HS3 and HS4. Genes Dev 9:3083, 1995[Abstract/Free Full Text]

14. Peterson KR, Clegg CH, Navas PA, Norton EJ, Kimbrough TG, Stamatoyannopoulos G: Effect of deletion of 5'HS3 or 5'HS2 of the human beta -globin locus control region on the developmental regulation of globin gene expression in beta -globin locus yeast artificial chromosome transgenic mice. Proc Natl Acad Sci USA 93:6605, 1996[Abstract/Free Full Text]

15. Enver T, Raich N, Ebens AJ, Papayannopoulou Th, Costantini F, Stamatoyannopoulos G: Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice. Nature 334:309, 1990

16. Behringer RR, Ryan TM, Palmiter RD, Brinster RL, Townes TM: Human gamma - to beta -globin gene switching in transgenic mice. Genes Dev 4:380, 1990[Abstract/Free Full Text]

17. Raich N, Papayannopoulou Th, Stamatoyannopoulos G, Enver T: Demonstration of a human epsilon -globin gene silencer with studies in transgenic mice. Blood 79:861, 1992[Abstract/Free Full Text]

18. Raich N, Clegg CH, Grofti J, Roméo P-H, Stamatoyannopoulos G: GATA1 and YY1 are developmental repressors of the human epsilon -globin gene. EMBO J 14:801, 1995[Medline] [Order article via Infotrieve]

19. Stamatoyannopoulos G, Josephson B, Zhang J-W, Li Q: Developmental regulation of human gamma -globin genes in transgenic mice. Mol Cell Biol 13:7636, 1993[Abstract/Free Full Text]

20. Ronchi A, Berry M, Raguz S, Imam A, Yannoutsos N, Ottolenghi S, Grosveld F, Dillon N: Role of the duplicated CCAAT box region in gamma -globin gene regulation and hereditary persistence of fetal haemoglobin. EMBO J 15:143, 1996[Medline] [Order article via Infotrieve]

21. Stamatoyannopoulos JA, Clegg CH, Li Q: Sheltering of gamma -globin expression from position effects requires both an upstream locus control region and a regulatory element 3' to the Agamma -globin gene. Mol Cell Biol 17:240, 1997[Abstract]

22. Roberts NA, Sloane-Stanley JA, Sharpe JA, Stanworth SJ, Wood WG: Globin gene switching in transgenic mice carrying HS2-globin gene constructs. Blood 89:713, 1997[Abstract/Free Full Text]

23. Liu Q, Bungert J, Engel JD: Mutation of gene-proximal regulatory elements disrupts human epsilon -, gamma -, and beta -globin expression in yeast artificial chromosome transgenic mice. Proc Natl Acad Sci USA 94:169, 1997[Abstract/Free Full Text]

24. Li Q, Clegg C, Peterson K, Shaw S, Raich N, Stamatoyannopoulos G: Binary transgenic mouse model for studying the trans control of globin gene switching: Evidence that GATA-1 is an in vivo repressor of human epsilon  gene expression. Proc Natl Acad Sci USA 94:2444, 1997[Abstract/Free Full Text]

25. Blom van Assendelft G, Hanscombe O, Grosveld F, Greaves DR: The beta -globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell 56:969, 1989[Medline] [Order article via Infotrieve]

26. Morley BJ, Abbott CA, Wood WG: Regulation of human fetal and adult globin genes in mouse erythroleukemia cells. Blood 78:1355, 1991[Abstract/Free Full Text]

27. Forrester WC, Novak U, Gelinas R, Groudine M: Molecular analysis of the human beta -globin locus activation region. Proc Natl Acad Sci USA 86:5439, 1989[Abstract/Free Full Text]

28. Wright S, Rosenthal A, Flavell R, Grosveld F: DNA sequences required for regulated expression of beta -globin genes in murine erythroleukemia cells. Cell 38:265, 1984[Medline] [Order article via Infotrieve]

29. Antoniou M, Grosveld F: beta -globin dominant control region interacts differently with distal and proximal promoter elements. Genes Dev 4:1007, 1990[Abstract/Free Full Text]

30. Hartzog GA, Myers RM: Discrimination among potential activators of the beta -globin CACCC element by correlation of binding and transcriptional properties. Mol Cell Biol 13:44, 1993[Abstract/Free Full Text]

31. Takegawa S, Brice M, Stamatoyannopoulos G, Papayannopoulou Th: Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids. Blood 68:1384, 1986[Abstract/Free Full Text]

32. Papayannopoulou Th, Brice M, Stamatoyannopoulos G: Analysis of human hemoglobin switching in MEL x human fetal erythroid cell hybrids. Cell 46:469, 1986[Medline] [Order article via Infotrieve]

33. Papayannopoulou Th, Enver T, Takegawa S, Anagnou NP, Stamatoyannopoulos G: Activation of developmentally mutated human globin genes by cell fusion. Science 242:1056, 1988[Abstract/Free Full Text]

34. Hanscombe O, Whyatt D, Fraser P, Yannoutos N, Greaves D, Dillon N, Grosveld F: Importance of globin gene order for correct developmental expression. Genes Dev 5:1387, 1991[Abstract/Free Full Text]

35. Peterson KR, Stamatoyannopoulos G: Role of gene order in developmental control of human gamma - and beta -globin gene expression. Mol Cell Biol 13:4836, 1993[Abstract/Free Full Text]

36. Dillon N, Grosveld F: Human gamma -globin genes silenced independently of other genes in the beta -globin locus. Nature 350:252, 1991[Medline] [Order article via Infotrieve]

37. Enver T, Ebens AJ, Forrester WC, Stamatoyannopoulos G: The human beta -globin locus activation region alters the developmental fate of a human fetal globin gene in transgenic mice. Proc Natl Acad Sci USA 86:7033, 1989[Abstract/Free Full Text]

38. Novak U, Harris EAS, Forrester W, Groudine M, Gelinas R: High-level beta -globin expression after retroviral transfer of locus activating region-containing human beta -globin gene derivatives into murine erythroleukemia cells. Proc Natl Acad Sci USA 87:3386, 1990[Abstract/Free Full Text]

39. Gelinas R, Novak U: Retroviral vectors for the beta -globin gene that demonstrate improved titer and expression. Ann NY Acad Sci 612:427, 1990[Abstract]

40. Chang JC, Liu D, Kan YW: A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression. Pr